The aim of this project is to investigate the role of proteins in regulating steroid synthesis, intracellular transport, steroid secretion, and steroid metabolism. Most of the work to date has dealt with specific adrenocortical steroid-protein interactions. However, the role of soluble proteins in regulating the mitochondrial cholesterol lyase system (rate-limiting step in steroidogenesis) has also been examined and experiments dealing with the role of proteins in steroid secretion have now been initiated. Several specific steroid-binding proteins have been discovered and partially characterized: one, the pregnenolone-binding protein, has now been purified and another, the pregnenolone sulfate-binding protein, has now been purified, and another, the pregnenolone sulfate-binding protein, has been partially purified. The pregnenolone-binding protein is a soluble protein of 34,000 MW whose binding activity is destroyed by heat, an acid environment, sulfhydryl reactants, and divalent cations. The pregnenolone sulfate-binding protein is a soluble protein which has a MW of 28-30,000. Currently, efforts are being made to produce antibodies to both of these proteins. The exact functional role of the binding proteins is not known as yet, and specific antibodies will be invaluable for examining this problem. It is of interest that both proteins bind products of the rate-limiting step in steroidogenesis. It should be noted, however, that the proteins also bind substrate and product of another enzyme system, i.e., pregnenolone sulfotransferase which also happens to be a soluble enzyme. Previously, this project has produced evidence for two classes of specific cholesterol-binding proteins in the adrenal cortex. Experiments eminating from this project are designed to yield insight into the molecular mechanisms of signal transduction following peptide hormone interaction at the cell surface, intracellular movement of steroid precussors, and secretion of steroid products. Since it is likely that proteins are mechanistically involved in these various vital functions, identification and purification of the specific proteins will be of considerable importance and should substantially advance our understanding of adrenocortical physiology and pathophysiology.